In Vitro-Evolved Peptides Mimic a Binding Motif of the G-Actin-Binding Protein Thymosin-B4 and Serve as Research Tools

Actin is the most abundant protein in eukaryotic cells and is
key to many cellular functions. Natural products that specifically recognize
the filamentous form of actin (F-actin) such as the bicyclic peptide phalloidin
are important tools to study actin and are widely applied for imaging the
cytoskeleton in cells. Herein, we aimed at developing peptide-based affinity
reagents that selectively bind to the monomeric form of actin (G-actin), for
which synthetic probes are not available. Panning a phage display library
comprising more than a trillion different bicyclic peptides against G-actin yielded
binders with low nanomolar affinity and greater than 1000-fold selectivity over
F-actin. Sequence analysis revealed a strong similarity of the peptides'
sequences to a region of thymosin-b4, a
protein that weakly binds G-actin, and competition binding experiments confirmed
a common binding region at the cleft between the subdomains 1 and 3 of actin. We
tested the G-actin peptides as probes in pull-down and imaging experiments and applied
a peptide variant with improved dissociation constant (Kd = 5 ± 2 nM) to measure the affinity
of G-actin-binding natural product toxins.